During the next year of support, we plan to continue to work towards fulfilling the specific aims, particularly the first three, listed in the original grant application. We plan to continue to isolate bronchial epithelial cells and to characterize them in terms of calcitonin (CT) production. Emphasis will be placed on further studies evaluating CT biosynthesis in the cultures of SCCL. In doing this, we intend to utilize some additional methods which will allow better characterization and thus more adequate comparison of the cultures in terms of their capability to produce calcitonin. In these studies, we will be isolating total cytoplasmic and poly A+ RNA from the different cell lines and evaluating them by RNA-DNA hybridization to determine the relative amount of calcitonin mRNA present within the various cultures. This then can be correlated with the secretion of calcitonin. If no messenger RNA is present, this would indicate that cells that are producing mRNA and those that are not differ at some level between transcription and translation, possibly at the level of gene activation. We plan to continue to evaluate the differences in the molecular forms of CT by chromatography, SDS-polyacrylamide gel electrophoresis, and utilization of newly developed immunoblotting techniques. The latter methods will entail extraction and concentration of CT molecules from CT-producing specimens using affinity gels we have recently prepared. Following electrophoresis and transfer of peptides from polyacrylamide gels to suitable nitrocellulose membranes, visualization of the resolved peptides will be by autoradiography of labeled antibodies or antibody: labeled-protein A complexes. It is postulated that there are constitutive, as well as stimulatable, pathways for hormone secretion in these cultures, and experiments will be carried out to test this hypothesis. It is postulated that the large molecular weight forms are produced largely through the constitutive pathway and the smaller molecular weight forms are secreted by the alternative pathway. Methods that may increase the production of hormone by one or another of these pathways will be evaluated. For example, it seems that acetylcholine will increase secretion of smaller molecular weight forms of CT and this may be through a stimulatory effect on this second pathway. (C)